The present invention relates to methods and to a device for depositing small amounts of liquid, for example for parallel analysis when comparing DNA sequences.
In the prior art, articles by Shena et al. (Science 270, 467-470, 1995) and by Cheny et al. (Nature Genetics Supplement 21, pp. 15-19, 1999) have described ways of depositing a hybridization microarray of on surfaces for a plurality of concurrent analysis operations running in parallel.
Small amounts of liquid have hitherto being deposited using one of the methods described briefly below. In the xe2x80x9cfountain penxe2x80x9d method, for example, spots of the liquid are applied to the surfaces by using a tool analogous to an ink pen. A disadvantage of this procedure that the amount of liquid deposited using this method depends very much on the surface tensions of the liquid both with respect to the tool and with respect to the surface. It is therefore difficult to control the amounts of liquid that are to be deposited. In general, although the wetting action of the liquid to be deposited is known, the interaction of the liquid with the tool and the surface has a non-negligible effect on the amount of liquid that finally adheres to the surface.
Another method of depositing liquids uses a very rapid-acting valve which briefly opens then re-closes a pressurized line in order to deposit a drop of liquid on a surface. It is, however, difficult to use this method for generating very high-density analysis arrays; the technical requirements for the rapid-acting valve that opens and re-closes the pressure line are exceptionally great, especially when the amount of liquid that is delivered needs to be controlled very accurately.
In the case of inkjet printers, it is common practice to use piezoelectric actuators. Although these can deliver very small amounts of liquid with a very high degree of accuracy, the procedure is technically very elaborate. Pipette tips with integrated piezoelectric actuators are extremely expensive and exceptionally fragile.
The disadvantage with the xe2x80x9cfountain penxe2x80x9d method briefly described above is the very large degree of fluctuation in the amounts of liquid deposited by means of this method. The essential disadvantage of the methods that use an extremely rapid-acting valve and the last method, which uses piezoelectric elements, is that a motorized dosing tip is needed for taking up the liquid to be pipetted. The technical complexity involved with this is exceptionally high, and a further problem is that the volume in the tube connection between the dosing tip and the pipetting head changes with temperature and as the tube moves.
On the basis of the prior art briefly described above, it is an object of the invention to reduce significantly the equipment outlay for a method of pipetting small amounts of liquid and to control accurately the amounts of liquid to be deposited by using the pipetting method, as well as to ensure that a high coverage density can be created on a substrate.
According to the invention, this object is achieved by the fact that, in a method of depositing small amounts of liquid on a substrate by using a pipette tip, which is connected to a flexible delivery line, liquid is taken up by expanding the volume in the delivery line and drops of liquid are deposited by exposing the line to an impulse that is transmitted to the liquid contained in it.
According to a device, which corresponds to the method of the invention, for depositing small amounts of liquid on a substrate by using a capillary, which is connected to a flexible delivery line, the flexible delivery line is provided with independently operated instruments for controlling its cross section, a hammer being provided for exerting an impulse on said line, and the hammer""s operating distance for changing the volume in the delivery line can be altered.
Many advantages can be achieved with the solution according to the invention. The invention allows accurately controllable, extremely small amounts of liquid to be deposited reproducibly and expediently on substrates and surfaces, without resorting to an expensive and mechanically very fragile piezoelectric pipette tip. The simple and economical pipetting head is easy to replace when necessary; after liquid has been taken up, a plurality of impulses can be transmitted to the delivery line so that a plurality of liquid drops can be deposited precisely in quick succession.
According to further refinements of the method of the invention, the instruments for controlling the cross section of the resilient delivery line are designed as compression valves which, for example, can be operated by electromagnetic means. One of these compression valves is closed while liquid is being taken up and is only opened to rinse the delivery line, whereas the other compression valve is opened to take up liquid so that the internal volume of the resilient delivery line is increased and liquid from a container can be taken up in it. In order to be dipped into the container, the pipette tip and the flexible delivery line may be held on a rail that can be moved up and down, allowing the pipette tip to be moved into the container or onto the substrate. The rail may be moved pneumatically, electromagnetically or using an electric motor.
The amount of liquid to be taken up may be metered by providing a variable stop on the cross-sectional control instrument that causes this dose to be taken up, the stop being used to vary the operating distance with which liquid is taken up. The amounts of liquid to be deposited are in the range of from 100 pl to 1 xcexcl, preferably in the range of from 500 pl to 10 nl, for which highly accurate and reproducible control of the drop size is absolutely necessary.
While the small amount of liquid is being deposited on the substrate, the flexible delivery line is closed at one end by a cross-sectional control instrument and an actuable hammer element, for example, is used to apply impulses to this line. The hammer may be operated electromagnetically or pneumatically, or actuation may be carried out mechanically using a spring with calibration by an electromagnet. In the region where the impulse is transmitted, the flexible delivery line, which is held inside a bore passing through a mount, is compressed by the impulse from the hammer to the extent of the latter""s preset operating distance and emits a liquid drop, the size of which depends on the change in volume due to the impulse, through the pipette tip onto the substrate. By means of a variable stop, which can be displaced by using adjustment means, it is possible to adjust the volume change which the impulse causes in the flexible delivery line.
After impulses that can be exerted have emptied the flexible delivery line, the compression valves can be opened so that a pressurized rinsing liquid can be fed through the flexible delivery line to the pipette tip in order to clean it. The pipetting head is subsequently moved to a washing position and then the rinsing medium is discharged.
The glass capillary having a very small outlet aperture, which can be used as the pipette tip, may be produced by heating one end of the capillary while using a pierced metal plate as a heat shield. The capillary is rotated while heating, so that its temperature can be increased uniformly until it softens. The surface tension of the glass causes the aperture of the capillary to contract during this procedure. The glass capillary may be coated fully or partially with a silane, for example, in order to obtain a specific surface tension.